This proposal is focused on the characterization of the global expression patterns of the Rickettsia typhus group rickettsiae. Members of the genus Rickettsia including R. prowazekii, the etiologic agents of epidemic typhus, are obligate intracellular bacterial parasites. We seek to understand the mechanisms by which rickettsiae cope with the problems and exploit the opportunities of the host cell cytoplasm, their unique and only niche. This is a niche that changes as the infection proceeds and as the host alternates from insect to mammal. R. prowazekii has only 834 genes. Our investigation will establish the gene-sets within these 834 genes that are expressed under changing environmental conditions by both members of the typhus group. The patterns of constitutive gene expression and regulated gene expression in response to environmental factors that are significant to the biology of rickettsiae will be determined. By coupling the recently completed genomic sequence of R. prowazekii and the advances in DNA Array technology with our expertise in transcriptional regulation and rickettsial biology, we can now characterize genetic expression in these obligate intracellular parasites in a global and comprehensive manner. Aim 1. Form a DNA Array on nylon filters containing all 834 genes of R. prowazekii Aim 2. Optimize the methods for synthesizing probes that will hybridize to the rickettsial genomic Array. Aim 3. Characterize the patterns of rickettsial genetic expression on a global and comprehensive scale. The total RNA will be extracted from infected host cells under conditions chosen to best demonstrate the regulatory diversity and adaptive biology of rickettsiae. The labeled-probes formed from all the rickettsial mRNAs present within a particular total RNA sample will be hybridized to the genomic Array of all R. prowazekii genes and analyzed to establish the complete repertoire of genes expressed under these conditions.